2.1. Drug Delivery
Drug delivery takes a variety of forms, depending on the agent to be delivered and the administration route. The most convenient way to administer drugs into the body is by oral administration. However, many drugs, in particular proteins and peptides, are poorly absorbed and unstable during passage through the gastrointestinal (G-I) tract. The administration of these drugs is generally performed through parenteral injection.
Although oral vaccination is more convenient, vaccines are generally given through injection. This is particularly true with killed or peptidic vaccines, because of their low absorbability and instability in the G-I tract. A problem with systemic immunization is that it may not effectively induce mucosal immune responses, particularly production of IgA, that are important as the first defense barrier to invaded microorganisms. For this reason, it would be beneficial to provide oral vaccination, if the problems of low absorbability and instability could be overcome.
Controlled release systems for drug delivery are often designed to administer drugs to specific areas of the body. In the gastrointestinal tract it is important that the drug not be eliminated before it has had a chance to exert a localized effect or to pass into the bloodstream.
Enteric coated formulations have been widely used for many years to protect drugs administered orally, as well as to delay release. Several microsphere formulations have been proposed as a means for oral drug delivery. For example, PCT/US90/06433 by Enzytech discloses the use of a hydrophobic protein, such as zein, to form microparticles; U.S. Pat. No. 4,976,968 to Steiner et al. discloses the use of "proteinoids" to form microparticles; and European Patent Application 0,333,523 by the UAB Research Foundation and Southern Research Institute discloses the use of synthetic polymers such as polylactic acid-glycolic acid to form microspheres.
Particles less than ten microns in diameter, such as the microparticles of EPA 0,333,523, can be taken up by cells in specialized areas, such as Peyer's patches and other intestinal mucosal lymphoid aggregates, located in the intestine, especially in the ileum, into the lymphatic circulation. Entrapping a drug or antigen in a microparticulate system can protect the drug or antigen from acidic and enzymatic degradation, yet still allow the drug or antigen to be administered orally, where they are taken up by the specialized uptake systems, and release the entrapped material in a sustained manner or are processed by phagocytic cells such as macrophages. When the entrapped material is a drug, elimination of the first-pass effect (metabolism by the liver) is highly advantageous.
2.2. Liposomes
Conventional liposomes have been proposed for use as an oral drug delivery system, for example, by Patel and Ryman, FEBS Letters 62(1), 60-63 (1976). Liposomes are typically less than 10 microns in diameter, and, if they were stable to passage through the G-I tract, may be absorbed through Peyer's patches (Aramaki, Y., H. Tomizawa, T. Hara, K. Yachi, H. Kikuchi, and S. Tsuchiya, 1993 Stability of liposomes in vitro and their uptake by rat Peyer's patches following oral administration. Pharm. Res. 10:1338, 1331; Childers, N., F. R. Donya, N. F. Magoo, and S. M. Michalek 1990. Ultrastructural study of liposome uptake by M cells of rat Peyer's patch: an oral vaccine system for delivery of purified antigen. Regional Immunology 3:8-16). Liposomes also have some features that should be advantageous for a particulate system for oral drug or antigen delivery. The phospholipid bilayer membrane of liposomes separates and protects entrapped materials in the inner aqueous core from the outside. Both water-soluble and -insoluble substances can be entrapped in different compartments, the aqueous core and bilayer membrane, respectively, of the same liposome. Chemical and physical interaction of these substances can be eliminated because the substances are in these different compartments. Further, liposomes are easy to prepare. However, liposomes are physically and chemically unstable, and rapidly leak entrapped material and degrade the vesicle structure. Without fortifying the liposomes, they are not good candidates for oral drug or antigen delivery. Thus, despite the early proposal for use of conventional liposomes in oral drug delivery, their use has still not been accepted.
Several methods have been tried to fortify liposomes. Some methods involve intercalating cholesterol into the bilayer membrane or generating the liposomes using phospholipids with high melting temperature or physically stabilizing preformed liposomes with excipients such as simple sugars or polysaccharides. Generally, these methods are not believed to be sufficient in making liposomes for oral delivery since during oral delivery liposomes are exposed to an acidic Ph in the stomach and bile salts and phospholipases in the intestine. These conditions typically dissolve the characteristic liposomal bilayer membrane and contents are released and degraded.
2.3. Polymerized Liposomes
Polymerization of liposomes has been shown in vitro to be an effective means of stabilizing the liposomes and reducing problems of degradation, agglomeration, and leakage of encapsulated drugs. Polymerized liposomes have been developed in attempts to improve oral delivery of encapsulated drugs (Chen et al., WO 9503035). The ability of polymerized liposomes to survive the G-I tract has also been investigated (Chen et al., 1995, Proceed. Internat. Symp. Control. Rel. Bioact. Mater. 22; Chen et al., 1995 Proc. 3rd U.S. Japan Symposium on Drug Delivery Systems; Brey, R. N., 1997, Proc. 4th U.S. Japan Symposium on Drug Delivery).
A number of compounds have been reported to form polymerized liposomes. For example, U.S. Pat. No. 4,248,829 discloses phospholipids containing di-yne acyl chains polymerizable by ultraviolet light to yield intermolecular or intramolecular cross-linking.
U.S. Pat. No. 4,485,045 discloses polymerizable phosphatidyl choline derivatives containing an unsaturated lower aliphatic acyloxy longer chain alkanoyloxy moiety. The polymerizable site in the phosphatidyl choline derivatives is a terminal ethylene group on the acyloxy substituent.
U.S. Pat. No. 4,808,480 discloses heterocyclic compounds containing disulfide bonds that are used to form polymerizable phospholipids. The phospholipids incorporate the heterocyclic disulfide compounds as terminal substituents on the glyceryl acyl groups, and polymerize upon ring-opening of the heterocyclic substituents.
U.S. Pat. No. 4,594,193 discloses polymerizable lipid compounds containing mercaptan groups. These lipids polymerize by formation of intermolecular disulfide linkages.
U.S. Pat. No. 5,160,740 discloses polymerization of a polymerizable 2,4-diene phospholipid, cholesterol, and a polymerizable 2,4-diene fatty acid to form a polymerized macromolecular endoplasmic reticulum. The reticulum is reported to be stable in surfactant solutions and capable of enclosing hemoglobin.
U.S. Pat. No. 5,466,467 discloses derivatives of phosphatidyl choline containing polymerizable acyl chain moieties and metal-chelating groups. The phospholipids contain iminodiacetic acid covalently bonded to the choline in the polar head group. Cross linked phospholipid membranes generated from monomeric units can be used to immobilize enzymes and proteins on the surface of the liposomes via metal bridges.
Further, U.S. Pat. No. 5,366,881 discloses phosphatidyl choline derivatives containing different polymerizable groups positioned at various sites in the acyl chains to achieve altered membrane fluidity properties. Additionally, the mixture of non-polymerizable phospholipids with polymerizable phospholipids provides for bilayer liposomes capable of conditional release of encapsulated material.
A number of additional polymerizable phospholipids are described in Regen, in Liposomes: from Biophysics to Therapeutics (Ostro, ed., 1987), Marcel Dekker, N.Y. Additional polymerizable moieties contained within the acyl chains of phospholipids or within the polar head group have been described and are found in Singh, A., and J. M. Schnur, 1993, "Polymerizable Phospholipids", in Phospholipids Handbook, Gregor Cevc, ed., Marcel Dekker, New York. Various other polymerizable phospholipids and fatty acids have been described, having methacrylate, vinylbenzene, diacetylenes, and azidoformaloxy groups within the structure of the acyl chains.
Although polymerized liposomes, generally, are more stable than their unpolymerized counterparts, it is not clear that the improved stability thus far achieved is by itself sufficient to enable these liposomes to deliver effective doses of drugs administered orally. Recent studies have investigated the possibility of modifying polymerized liposomes to contain a molecule or ligand which selectively targets M cells and other absorptive cells in the mammalian intestine (Chen et al., 1996, Pharmaceutical Research 13:1378-1383). Incorporation of a targeting ligand is believed to increase the adhesion efficiency of the modified polymerized liposome on M cell surfaces, and thus to increase the efficiency of absorption of drugs encapsulated in those liposomes. M cells are specialized epithelial cells dispersed within the follicle associated epithelium (FAE) overlying the Peyer's patches in mammalian small intestine. The use of targeting ligands specific for surface receptors on M cells, enterocytes or other cells requires new chemistries to effectively incorporate such ligands without compromising the stability or safety of the polymerized liposome.
A variety of methods have been described for covalently coupling of bioactive ligands to the surface of conventional liposomes. U.S. Pat. No. 5,171,578 discloses the chemical coupling of the glycoprotein streptavidin to the surface of liposomes via a modified phosphatidyl ethanolamine. Because of selective binding affinity to biotin, such surface modified liposomes can be used to directly bind biotinylated proteins to their surface.
U.S. Pat. No. 5,204,096 describes the covalent coupling of peptides to the surface of liposomes by activating peptides with carbodiimide followed by coupling to active carboxyl groups exposed on the surface of liposomes. In this case, surface carboxyl groups are provided by the inclusion of aminoalkanes, such as stearylamine or diamino alkanes in the lipid bilayer.
U.S. Pat. No. 5,258,499 discloses the preparation of a liposome cytokine complex in which the procedure for covalent attachment of receptor-binding interleukin-2 involves treatment of the cytokine with succinimidyl-4-(p-maleimidophenyl)butyrate as a linker followed by linkage to activated liposome surfaces. In this case, the activated liposome surface consists of phosphatidyl ethanolamine modified with succinimidyl-S-acetylthioacetate.
Zalipsky et al (Zalipsky, S., Mullah, N., Harding, J. A., Gittelman, J., Guo, L. and DeFrees, S. A., 1997, Bioconjug. Chem. 8:111-118) described the synthesis of a lipid anchor for the surface modification of liposomes, containing distearoylphosphatidylethanolamine (DSPE) as a lipid anchor, heterobifunctional polyethylene glycol (PEG) with a molecular weight of 2000 as a linking moiety, and biological cell adhesive ligand [YIGSR peptide or Sialyl Lewis (X) oligosaccharide (SLX)]. Allen et al (Allen, T. M., Brandeis, E., Hansen, C. B. Kao, G. Y. Zalipsky, S. 1995. Biochim Biophys Acta. 1237:99-108) described the derivitization of the surface of sterically stabilized liposomes. The polyethylene glycol (PEG)-lipid derivative pyridylthiopropionoylamino-PEG-distearoylphosphatidylethanolamine (PDP-PEG-DSPE) was synthesized and incorporated into liposomes. Thiolysis of the PDP groups resulted in formation of reactive thiol groups on the liposome surface which reacted with maleimide-activated antibodies to yield covalent attachment of the antibodies. Kirpotin et al (Kirpotin, D., Park, J. M., Hong, K., Zalipsky, S., Li, W. L., Carter, P., Benz, C.C., Papahadjopoulos, D. 1997. Biochemistry 36:66-75) described the formation of liposomes conjugated via PEG-modified distearoylphosphatidyl phosphatidylethanolamine to Fab fragments of a humanized recombinant Mab against the extracellular domain of the breast cancer marker HER2/neu by maleimide-terminated membrane-anchored spacers of two kinds for covalent attachment at the distal terminus of the PEG chain.
2.4. Lectin Targeting Of Liposomes
Lectins have been proposed as promising moieties to use as targeting ligands. Lectins are a broad group of proteins, usually glycoproteins of plant origin, with binding specificity for particular carbohydrates. Like any targeting ligand, lectins can be covalently bound to the lipids of the liposome, or can be non-covalently attached to the liposome by a combination of short-range intermolecular forces and simple steric entanglement. Surface-bound lectins can aid in the selective targeting of liposomes with entrapped drug or antigen to carbohydrate counter-ligands expressed on cell receptors or other surface glycoproteins. In order to effectively target particular cells, the lectins must be attached to the liposome so that the site-specific portion of the lectin is exposed and available for binding to cells. Additionally, the targeting lectin must be incorporated into the liposome in a manner that does not destabilize the liposome or allow physical release of the targeting ligand from the liposome surface. A wide variety of lectins with selectivity to intestinal absorptive cells and M cells has been identified (Gianasca, P. J., K. T. Gianasca, P. Falk, J. I. Gordon, and M. R. Neutra 1994. Gastrointen. Liver Physiol. 30:G1108-1121; Clark, M. A., M. A. Jepson, and B. H. Hirst 1995. Lectin binding defines and differentiates M-cells in mouse small intestine and caecum. Histochem Cell Biol. 104:161-168). Recent work has shown that lectins with selectivity to intestinal M cells and enterocytes can be incorporated into liposome bilayers and the liposomes subsequently polymerized (Chen et al., 1996, Pharmaceutical Research 13:1378-83). These lectin-modified polymerized liposomes show increased efficacy in targeting liposomes to Peyer's patches in the G-I tract. However, the lectin-modified anchoring lipids used in these studies were not structurally optimized for stability within the polymerized liposome bilayer and formed patches of non-polymerized lipids that contributed to instability.
Despite the advances in liposome technology and drug delivery, there remains a need for stable and efficacious polymerized liposomes, and new polymerizable compounds that can be incorporated into polymerizable liposomes to improve stability, binding selectivity, and efficiency of drug delivery. There additionally remains a need for new processes to manufacture polymerizable liposomes incorporating targeting molecules or ligands, and to manufacture polymerizable liposomes which encapsulate drugs.